Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8
Fig 4
IpaH9.8 mutants display decreased activity to degrade the GBPs.
(a) IpaH9.8 mutants show reduced interaction with the GBPs. Flag tagged GBPs were co-expressed with 6xMyc tagged IpaH-C337A in HEK293T cells. The GBPs were immunoprecipitated from the cell lysates using the Flag-M2 beads, and associated IpaH was detected by immunoblotting. (b) Degradation of RFP-GBP1 by different IpaH9.8 mutants. HeLa cells stably expressing RFP-GBP1 and scFV-Suntag-GFP were infected with S. flexneri ΔipaH9.8 expressing various IpaH9.8 proteins fused to 10xSunTags. In uninfected cells, GCN4-GFP (green) displays a dispersed pattern in the cell. Infection of S. flexneri expressing wild-type IpaH9.8-10xSunTag leads to the enrichment of GFP signal in the cytoplasm, due to the delivery of IpaH9.8 by the bacteria and recognition of the SunTags by GCN4. The RFP signal (red) is largely diminished due to the degradation of GBP1 by IpaH9.8. In contrast, IpaH mutants have impaired functions to degraded GBP1, resulting in the recruitment of RFP-GBP1 onto the bacteria. Nuclei were stained with DAPI (blue). The data are representatives of two independent experiments.